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. Author manuscript; available in PMC: 2016 Mar 17.
Published in final edited form as: Sci Transl Med. 2012 Feb 29;4(123):123ra24. doi: 10.1126/scitranslmed.3003500

Fig. 1.

Fig. 1

Effect of c-Abl1 knockdown and kinase inhibition on Ebola VLP release in transfected 293T cells. (A) Knockdown of c-Abl1 (lanes 1 to 3) or c-Abl2 (lanes 4 to 6) using nontargeting siRNA control or siRNA targeting c-Abl1 or c-Abl2 confirmed by Western analysis in cell lysates with antibodies specific to either c-Abl1 (lanes 1 to 3) or c-Abl2 (lanes 4 to 6). β-Actin was used as a loading control. The results are representative of five independent experiments. 293T cells were transfected with plasmids encoding VP24, VP35, VP40, NP, and GP. In all cases, Ebola VLPs were analyzed by immunoprecipitation with GP followed by Western blotting for NP and VP40 (lanes 10 to 12). Cell lysates are shown in lanes 7 to 9. Data represent means ± SEM of individual measures with cells from four independent experiments. Significant differences by paired Student’s t test between test and control siRNAs are indicated. *P < 0.05. (B) Knockdown of c-Abl1 using a nontargeted siRNA control (lane 13) or three individual siRNAs (S9, S10, and S11) targeting c-Abl1 (lanes 14 to 16) was analyzed by Western analysis in cell lysates, with eIF4E as a loading control. NP and VP40 present in cell lysates were determined (lanes 17 to 20), and Ebola VLPs in supernatants were measured as in (A) after knockdown with c-Abl1 individual siRNAs (lanes 21 to 24). Data represent means ± SEM of individual measures with cells from three independent experiments. Significant differences by paired Student’s t test between test and control siRNAs are indicated. *P < 0.05. For (A) and (B), quantitation of NP and VP40 protein bands is expressed as a percentage of the intensity of the siRNA control band (lower panels). (C) Western analysis of Ebola VLP release for NP and VP40 content with imatinib (lanes 25 to 27) or nilotinib (lanes 28 to 30). Water was used as control for imatinib and DMSO for nilotinib. Quantitation of NP and VP40 was performed relative to the intensity of the solution control band. Data are presented as means ± SEM of individual measures with cells from eight (for NP) and three (for VP40) independent experiments. Significant differences by paired Student’s t test between drug and vehicle control are indicated. *P < 0.05.